Bioluminescence is a phenomenon based on a chemical reaction in vivo, which is called a luciferin (a luminescence substrate)-luciferase (an enzyme that catalyzes the luminescence reaction) reaction. Numerous studies of the identification of luciferins or luciferases and the elucidation of the luminescence mechanism in a molecular level have been performed for a long time in the country and overseas. In bioluminescent marine organisms, Oplophorus gracilirostris luciferase from the deep-sea shrimp is an extracellularly secreted luciferase (Non-Patent Document 1). Oplophorus luciferase is a 106 kDa protein composed of a protein with a molecular weight of 35 kDa and a protein with a molecular weight of 19 kDa. The domain that catalyzes the luminescence reaction is found to be 19 kDa protein. Oplophorus luciferase uses coelenterazine as a luminescence substrate and is classified as a coelenterazine-type luciferase (Patent Document 1, Non-Patent Document 2). Oplophorus luciferase is different from other coelenterazine-type luciferases in broad substrate specificity and uses coelenterazine analogues as a suitable substrate as well as coelenterazine (Non-Patent Document 2). When the gene for the 19 kDa protein is expressed in Escherichia coli (E. coli) at ordinary and lower temperatures, the protein is expressed mostly as an insoluble protein (Non-Patent Document 3). When the 19 kDa protein was expressed as a fusion protein to ZZ domain from protein A in a low temperature expression system, the fused protein could be expressed as a soluble protein (Non-Patent Document 4). It is reported that when the 19 kDa protein was expressed in animal cultured cells, the expressed protein was hardly secreted outside of cells (Non-Patent Document 2).
Recently, it is reported that the mutated 19 kDa protein having catalytic activity of luminescence was prepared by mutating the 16 amino acids of the 19 kDa protein and showed higher luminescence activity than native 19 kDa protein, and was secreted into an extracellular medium (Patent Document 2, Non-Patent Documents 4 and 5). It is also reported that coelenterazine derivatives displayed higher activity than native coelenterazine used as a substrate (Non-Patent Documents 4 and 5).
In the luminescence reaction system using coelenterazine as a substrate for the luminescence reaction, the luminescence reaction of luciferasae proceeds only by a substrate and molecular oxygen. For this reason, a coelenterazine-type luciferase gene is used widely as a reporter assay in an animal cultured cell system at present. Renilla luciferase having 311 amino acids is used for a reporter assay inside of cells. For an extracellular reporter assay, the secretory Gaussia luciferase having 168 amino acids is used. Comparison in specific activity between recombinant Renilla luciferase and recombinant Gaussia luciferase using coelenterazine as a substrate reveals that the specific activity of Renilla luciferase is about 1/100 of Gaussia luciferase (Non-Patent Documents 5 and 6). On the other hand, the specific activity of the mutated 19 kDa protein which catalyzes the luminescence reaction is 1/10 of Gaussia luciferase and the mutated 19 kDa protein was found to be obviously inferior as a reporter assay gene as a secretory protein by comparison with Gaussia luciferase.
In view of the foregoing, there has been desired a reporter gene that is a luciferase expressed intracellularly and exhibits higher luminescence activity than wild 19 kDa protein, when not only coelenterazine but its analogue is used as a substrate.